This combination enhanced the robustness of the relative quantification analyses. These findings highlight the importance of validating reference genes before quantifying target miRNAs. Furthermore, our findings will improve studies that monitor hepatitis progression and will aid in the discovery of noninvasive biomarkers to diagnose early stage HCC.Serum exosomal microRNAs (miRNAs) have received considerable attention as potential biomarkers for diagnosing cancer. The canonical technique for measuring miRNA transcript levels is reverse transcription quantitative polymerase chain reaction (RT-qPCR). One prerequisite for validating RT-qPCR data is proper normalization with respect to stably expressed endogenous reference genes. However, genes that meet all of the criteria of a control gene for exosomal miRNAs have not yet been identified. To find out the control gene for exosomal miRNAs, we evaluated the expression stability of 11 well-known reference genes in circulating exosomes. In this study, we found that the combination of  Exosomes are 40–100 nm diameter membrane-bound microvesicles of endocytic origin that are released from different cell types under both normal and pathological conditions. Exosomes have been identified in body fluids such as urine, amniotic fluid, malignant ascites, saliva, and blood . Exosome miR-21 expression is useful for diagnosing HCC [It has been reported that the majority of serum miRNAs are enriched in exosomes. Exosomal miRNAs could serve as valuable noninvasive biomarkers for distinguishing the type and grade of liver inflammation . Additiosing HCC .Currently, stem-loop reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is widely used to quantitatively analyze circulating miRNAs . More imPrevious studies have demonstrated that none of the commonly used reference genes for normalization are universal for all tissue types or experimental situations –13. SuitThe Medicine Ethics Committee of Second Affiliated Hospital of the Third Military Medical University approved all aspects of this study in accordance with the Helsinki Declaration. All the patients or their guardians provided written informed consent.Preoperative blood from 50 HCC patients was collected at the Second Affiliated Hospital of the Third Military Medical University . Blood was collected from 50 CHB patients without HCC and 50 healthy subjects prior to antiviral treatment at the Second Affiliated Hospital of the Third Military Medical University . A biopsy was performed to diagnose HCC. Chronic HBV infection was defined as the persistence of HBV surface antigen in the bloodstream for at least 6 months. Healthy controls who were not infected with HBV, HCV, or HIV, had normal liver function tests, and had no history of liver disease were selected. The clinical characteristics of the study participants are presented in All samples were taken from January to August 2014. The peripheral blood samples were collected in 5 mL Vacutainer SST Plus Blood Collection Tubes . Samples were incubated at room temperature for between 30 min and 2 h. The tubes were centrifuged at 1,500 g for 10 min, and the serum samples were aliquoted and centrifuged again at 2,000 g to completely remove any remaining cells. The serum samples were stored at −80°C until further processing for exosome isolation.µL of ExoQuick exosome precipitation solution. Exosome isolation was performed according to the manufacturer's protocol . Briefly, the samples were incubated at 4°C for 30 min and then centrifuged at 13,000 rpm for 2 min. The protein-rich supernatant was removed, and the exosome-rich pellet was retained for RNA extraction or Western blot.Two hundred and fifty microliters of serum was mixed with 66 Electron microscopy was performed on serum exosome samples at the Biomedical Analysis Center, Third Military Medical University, Chongqing, China. Samples were prepared as described by Thery et al. . BrieflyThe exosome-rich pellet and a human hepatocellular carcinoma Huh-7 whole cell extract (control) were resuspended in 1X RIPA buffer, separated on a polyacrylamide gel, and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with bovine serum albumin and incubated first with a CD63 antibody  or a CD9 antibody  and then with a goat anti-rabbit HRP secondary antibody . The proteins were detected by enhanced chemiluminescence .2. When the cells reached approximately 90% confluence , they were collected and washed three times with 1X PBS.Huh-7 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 g/mL streptomycin at 37°C with 5% COmiR-26a, miR-221, miR-22*, miR-181a, miR-181c, miR-16, miR-103, miR-191, and let-7a) and two small RNAs (5SrRNA and U6snRNA) were selected as candidate reference genes to normalize the miRNA RT-qPCR data [http://dx.doi.org/10.1155/2015/893594). Primers for 5SrRNA and U6snRNA were purchased from RiboBio . The NCBI (http://www.ncbi.nlm.nih.gov/) and miRBase (http://www.mirbase.org/) databases were used to search for available gene sequences, and Primer5 software was used to design the primers. The reaction conditions were optimized by determining the optimal annealing temperature and primer concentration.Nine miRNAs . RNA was isolated using a miRNeasy Serum/Plasma Kit according to the manufacturer's protocol for liquid samples . Each RNA sample was eluted in the same volume  after extraction from a given volume of serum (250 μL) and reverse transcribed to cDNA using the GoScript Reverse Transcription System .Exosome-rich pellets were resuspended in 200 µL, which included 10 µL of SYBR Select Master Mix , 250 nM of each primer, and 2 µL of a 1 : 5 dilution of cDNA. The following thermal cycling conditions were utilized: 1 cycle at 50°C for 2 min, 1 cycle at 95°C for 2 min, and 40 cycles of amplification at 95°C for 15 s and 60°C for 1 min. The threshold cycle (Ct) was calculated using Applied Biosystems SDS software 2.1 .Quantitative PCR was performed in 96-well reaction plates with a StepOne Plus Real-Time PCR System . The final reaction volume was 20 (E) Standard curves were generated to calculate the RT-qPCR efficiency using 10-fold serial dilutions from a cDNA pool [R) and PCR efficiency (E) for each assay were determined from the respective plots. The R2 and E values were calculated using GenEx Standard software . The amplification efficiency was calculated using the following formula: efficiency (%) = (E − 1) × 100. In this study, the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines [DNA pool . Duplicaidelines  were folM). Genes with the lowest M are the most stably expressed, whereas the highest M values indicate the least stable expression. An M value of ≤1.5 indicates a stably expressed gene. To determine the optimal number of control genes for normalization, the pairwise variation, Vn/n + 1, was calculated as the geometric mean of the relative expression values of the reference genes in the different samples based on the normalization factors (NFn and NFn+1) [Vn/n + 1 values of less than 0.15 for proper normalization, this requirement is not absolute. Other studies have reported no significant improvement with Vn/n + 1 values exceeding 0.2 [The geNorm and NormFinder algorithms were used to calculate the expression stabilities of the candidate reference genes. The geNorm algorithm ranks the tested genes based on their stability measure (d NFn+1) . Althougding 0.2 . NormFinding 0.2 . In thisn reference genes and is calculated as the geometric average of the n raw reference gene quantities for any given gene i.Here, the Acc.S.D. is based on 2-transformed for analysis. The distribution of continuous data was determined using the Kolmogorov-Smirnov test, and t-tests were used to identify significant differences in target miRNA expression between two groups. If these data were not normally distributed, we used nonparametric hypothesis tests to determine their distribution. The statistical tests were two-sided, and P values of less than 0.05 were considered statistically significant. The data were analyzed using SPSS 19.0 .GenEx Standard software was used to calculate the expression of target miRNAs relative to suitable normalization genes. All the data were corrected based on their respective amplification efficiencies, and the relative quantification (RQ) values were calculated for all the samples within each group. Next, the RQ values in the each group were adjusted to the maximum value and logThe successful isolation of exosomes from serum is necessary to accurately quantify exosomal miRNA. In our study, we obtained serum exosomes using ExoQuick exosome precipitation solution. Under an electron microscope, exosomes are small membrane-bound vesicles with diameters ranging from 40 to 100 nm . The exomiR-16, miR-103, miR-22*, miR-26a, miR-221, miR-181a, miR-181c, let-7a, and miR-191) and two small RNAs (5SrRNA and U6snRNA) [The optimal reference gene(s) were identified from among nine stable miRNAs (U6snRNA) , 16 thatE%) was calculated from the slope of the standard curve. The E values for the 11 reference genes varied from 91.9% to 98.2%, and the regression correlation coefficients (R2) ranged from 0.988 to 0.999 (Table S2). The melting curves exhibited single peaks corresponding to unique amplicons.We subsequently determined the reliability of quantifying these candidate genes. The gene-specific PCR amplification efficiency . Therefore, geNorm recommended six miRNAs  (RG-6) as the optimal combination of reference genes  was selected by geNormas as the least variable among all the reference genes  (RG-7)  (RG-7) . let-7a and miR-221, and let-7a was also considered to be the least variable gene by NormFinder . This combination was also the most stably expressed in all the samples. In contrast to geNorm, NormFinder considered miR-103 to be less stable and recommended the other five genes (RG-5) as the optimal reference gene set.HCC patients and healthy persons were grouped in the second subset. The most stable genes based on the geNorm analysis were Figures . 5S and  miR-221, miR-103, let-7a, miR-191, miR-26a, and miR-181a (RG-6d) as the least variable set of reference genes  as the optimal reference gene set  or U6 (CCG-1) was used as reference gene, there was no significant difference in miR-21 expression between patients with HCC and CHB  and endogenous control genes. External spike-in controls are not assumption-free; it is assumed that the experimenter starts with the same quantity of equal quality template. Normalization factors that are based on endogenous miRNA are therefore preferred.In this study, we selected optimal reference genes in four steps to improve the accuracy with which differences in serum exosomal miRNAs between patients with liver disease and healthy controls can be ascertained . To the  miR-221, miR-103, let-7a, miR-181c, miR-181a, and miR-26a (RG-6) were the most stably expressed reference genes in all the samples. Among the top reference genes, miR-221 was identified as the most stable, followed by miR-103 and let-7a. Similar results were observed by Qi et al., who found that miR-221 was not differentially expressed in the sera of HCC patients and healthy subjects [ miR-103 ranked as the most stably expressed reference gene for acetaminophen hepatotoxicity in a study by Wang et al. [ let-7a was predominantly exosome-associated and might be of cellular origin [ let-7a was ranked as the third most stable reference gene. Similarly, let-7a ranked among the top most stable reference genes in a breast cancer study [ miR-26a was the most stable miRNA in porcine liver and that the combination of miR-26a, let-7a, and miR-103 was recommended as the optimal reference gene set for liver studies [Currently, the majority of HCC is thought to be a consequence of chronic liver inflammation. Viral and host inflammation-related factors are important predictors of HCC prognosis after surgery . We meassubjects . Additior origin . In our er study . Timoned studies .M value predominantly based on the intragroup variation, so it must exclude coregulated or coexpressed genes. This discrepancy has been previously described [Our study confirmed the importance of reference gene optimization for each RT-qPCR experiment. Different experimental settings can result in changes in the stability of the reference genes. In our study, all the candidate reference genes were analyzed in three subsets using geNorm and NormFinder. We identified five different reference gene combinations. There are two brief reasons for this distinction. First, the NormFinder program estimates both the intra- and intergroup variation and combines them into the stability value. However, geNorm calculates an escribed , 19, 30.escribed , 12, 28. miR-21. Except for RG-2, other combinations of reference genes recommended by NormFinder or geNorm were propitious to assess the biological variability of target miRNAs ( miR-181c (RG-1) as a control gene. Although there have been many reports on screening miRNA expression in serum samples or tissue samples from patients with liver disease by PCR array analysis, none of these studies identified a significant change in miR-181c in hepatopathy samples. In our study, miR-181c was considered to be a stable reference gene. However, we found that miR-181c introduced bias into the analysis and led to the misinterpretation of miR-21 expression in HCC patients compared with CHB patients.The practicality of a normalization gene also influences the selection of an optimal reference gene. Therefore, we tested the suitability of the different approaches witht miRNAs . It was  U6 (CCG-1) for the relative quantification of target miRNAs. Our results indicated that the normalization of exosomal miRNA expression using CCG-1 was inappropriate. Compared with the results of Wang et al.'s study [ miR-21 expression in HCC than in CHB when using the same reference gene U6 (CCG-1). This discrepancy may be due to subject selection. We demonstrated wide differences in the expression of exosomal U6 in our study. This unstable expression led to differential results when U6 was used as a normalization factor for miR-21.We also selected another common housekeeper gene like's study , we did  miR-21 expression between CHB patients and HCC patients. Therefore, for comprehensive investigation into the progression of CHB to HCC, we considered that the combination of miR-221, let-7a, miR-191, miR-26a, and miR-181a (RG-5d) was the optimal reference gene set, on account of the technical and economic advantages of using a smaller number of reference genes. Furthermore, it was inappropriate to normalize the data with U6 or miR-181c. The present study, which identified optimal reference genes, will improve studies that monitor the progression of hepatitis and will help identify noninvasive biomarkers to diagnose early stage hepatic carcinoma.In summary, we presented the first experimentally validated optimal reference genes to normalize miRNA expression in serum-derived exosomes. All of the combinations proposed in this study were appropriate for normalization. However, RG-2was not reliable as a reference gene across the groups, especially when used to normalize target genes with smaller fold changes. RG-5 had lower sensitivity for the comparability ofSupplemental Table 1 shows The primer sequences for the candidate reference genes, along with their corresponding accession numbers and each of Real-Time PCR Experiments amplification efficiency.Supplemental Table 2 shows our RT-qPCR analyses followed MIQE guidelines. These guidelines promote experimental consistency and transparency and increase the reliability of the obtained results.


Bioinformatics demonstrated that variant rs17168525 was identified to be located in the let-7/miR-98-binding site of myotrophin. We further experimentally test to effects of the identified variant on myotrophin translation using luciferase reporter assay and Western blotting. We found that the C allele of rs17168525 suppressed myotrophin translation by facilitating let-7c binding, but not the T allele. Let-7c overexpression caused a significant decrease in the level of myotrophin protein. Next, we investigated the association of the variant with cardiac hypertrophy in 1614 hypertensive patients, including 552 with left ventricular hypertrophy and 1062 without left ventricular hypertrophy, as well as 591 healthy control subjects from a Han Chinese population. No significant association between the variant rs17168525 and left ventricular hypertrophy in hypertensive patients in a Han Chinese population (P>0.05). In conclusion, our experimental results provide evidence that the T allele of rs17168525 in the 3′-UTR of myotrophin might influence the level of myotrophin protein by interfering with let-7/miR-98 binding.Myotrophin, known as a myocardial hypertrophy-inducing factor, is responsible for the initiation of cardiac hypertrophy that transits to heart failure. MicroRNAs are small noncoding RNAs that down-regulate posttranscriptional expression of target molecules. We investigated the role of variants of the microRNA-binding site in  Cardiac hypertrophy is recognized as an adaptive process to a variety of physiological and pathological conditions . ProlongIn vivo and in vitro studies have shown that myotrophin plays a vital role in the initiation of cardiac hypertrophy that transits to heart failure [Myotrophin, a 12-kDa protein, is identified in spontaneously hypertensive rat hearts and cardiomyopathic human hearts . In vivo failure –10. The  failure  and thosangiopoietin-1 might reduce the risk of stroke by interfering with miR-211 binding efficiency [Emerging evidence has indicated the importance of microRNAs (miRNAs) in the regulation of hypertrophic process –16. MiRNficiency .myotrophin. In the present study, we aimed to investigate whether the rs17168525 can modify the efficiency of let-7/miR-98 binding to myotrophin and then further contribute to the genetic susceptibility to cardiac hypertrophy.Through a bioinformatics approach, we identified a variant, rs17168525, located in the let-7/miR-98 target site in the 3′-UTR of myotrophin 3′-UTR-luciferase reporter plasmids, a 46 bp sequence  were verified by sequencing.To construct 5 cells/well) were co-transfected with 2 μg of myotrophin-pMIR-C or myotrophin-pMIR-T plasmid, 0.01 μg of Renilla luciferase and 50 pmol of PremiR miRNA precursor of let-7c or PremiR Negative Control (Ambion), all combined with Lipofectamine 2000 . 48 h after transfection, the cells were washed and lysed with Passive Lysis Buffer , and the luciferase activities were measured using a luminometer .Hela cells . Neonatal rat ventricular myocytes were isolated and cultured from the ventricles of 1- to 2-day-old rats. In brief, left ventricular myocytes were dispersed from the ventricles by digestion with HEPES-buffered saline solution containing 0.06% collagenase. And then, cells were plated in DMEM with 10% fetal bovine serum, 0.1 mM bromodeoxyuridine (BrdU) at a density of 106 cells/ml. After transfection of PremiR miRNA precursor or Anti-miR miRNA inhibitor of let-7c for 48 h, the cells were lysed with Cell Lysis Buffer. We detected let-7c expression levels by using Taqman probe-based real-time PCR after treatment with PremiR miRNA precursor or Anti-miR miRNA inhibitor of let-7c. Equal amounts of proteins (5–30 μg) were subjected to SDS-PAGE and transferred to nitrocellulose membrane. The membrane was incubated with myotrophin antibody (Santa Cruz) or β-tublin  at 4°C overnight. Fluorescence-conjugated rabbit secondary monoclonal antibody  was added and incubated at room temperature for 1 h. Proteins were detected using the AP-NBT/BCIP system.Cardiomyocytes were plated at a concentration of 10Subjects were recruited from 24 communities in Xinyang, Henan Province, People’s Republic of China. The study was started in April 2005. All studies were approved by the Ethics Committee of Fuwai hospital. Signed informed consent was obtained from all participants, following a protocol that had been approved by the Ethics Committee of Fuwai hospital, and the study was conducted in accordance with the Declaration of Helsinki. The subjects consisted of 552 hypertensive patients (mean age 59.9±7.4 years) with LVH and 1062 patients (mean age 57.5±8.4 years) without LVH . HyperteBlood samples were taken after a 12 h overnight fasting. Plasma and urine biochemical variables were determined by using standard methods with an automatic analyzer . All of the participants completed a questionnaire on current medication and life style. A complete medical history was obtained from all subjects, including family history of hypertension, diabetes mellitus and the following cardiovascular risk factors: alcohol intake, cigarette smoking, family history, weight, height, BMI (body mass index), SBP and DBP. All measurements and echocardiography were performed as described previously .myotrophin. Genomic DNA was extracted from peripheral blood using a method described previously [2+, 2 μl of dNTPs, 0.3 μl of Taq polymerase, 4 μl of 1× Q-solution, 0.4 μl of primer mix and 9.7 μl of ddH2O. The PCR included initial denaturing at 95°C for 10 min, followed by 35 cycles of denaturing at 94°C for 30 s, annealing at 59°C for 90 s, and extension at 72°C for 1 min. The reaction was completed by a final extension at 72°C for 7 min. Reactions were performed on a thermal cycler Gene Amp PCR system 9600 . Further amplification was performed in a 10 μl volume of multiplex LDR reaction mixture, containing 1 μl (100 ng) of the resultant probe mix, 1 μl of probe mix, 0.05 μl of NEB Taq DNA ligase and 7.95 μl of ddH2O. The LDR conditions included initial denaturing at 95°C for 2 min, followed by 35 cycles of denaturing at 94°C for 30 s and annealing at 50°C for 2 min. LDR products (1 μl) were mixed with 1 μl of ROX  and 1 μl of loading buffer, detected in an ABI PRISM 377 DNA Sequencer, and analyzed with Genemapper . Reproducibility of genotyping was confirmed by sequencing in 400 randomly selected samples with 100% concordance.The variant rs17168525 was selected based on its relevance to gene expression, being located in the let-7/miR-98 target site of the 3′-UTR of eviously . The var2 test was used to test categorical variables, and the Hardy—Weinberg equilibrium was used to test the variant frequencies. One-way ANOVA was used for comparison of quantitative variables. Differences of quantitative variables between groups were analyzed using the Student′s t-test. ORs (odds ratios) and 95% CIs  were calculated by use of multivariate logistic regression analyses, and were adjusted by age, gender, BMI, SBP, DBP, smoking status, alcohol consumption and glucose. Statistical analysis was performed with the SPSS 13.0 package. All significant tests were two-tailed and were considered significant at P<0.05.Values are expressed as means ± S.D. A χhttp://pictar.bio.nyu.edu.) to screen potential miRNAs target sites in myotrophin, and discovered that variant rs17168525 occurs in the middle of the let-7/miR-98 complementarity seed binding sequence  and mutant sequence  of rs17168525, respectively. It should be noted that miR-98 and some other members of the let-7 family have a very similar, if not identical, seed sequence and share target genes   . This dein vitro luciferase activity study.To examine whether let-7c down-regulates myotrophin in cardiomyocytes, we conducted western blotting to analyze myotrophin protein levels in primary neonatal rat cardiomyocytes with transfection of Pre-let-7c or Anti-miR miRNA inhibitor of let-7c (Anti-let-7c). Pre-let-7c increased the expression of let-7c Fig A in . LikewisTo test the hypothesis that the variant rs17168525 would modify genetic susceptibility, we investigated the association of variant rs17168525 with cardiac hypertrophy in a case-control study. The clinical characteristics of subjects are shown in 2 test in hypertensive patients with LVH compared with either hypertensive patients without LVH or controls in either genotype frequency distribution . In addition, the association between the variant rs17168525 and LVH was not significant in either men or women  of rs17168525 was 0.008 for CEPH, 0.089 for JPT and 0.189 for Han Chinese in Beijing. Consistently, our present study showed that the MAF of rs17168525T allele was 0.179 for Han Chinese in Xinyang region, but higher than that registered for CEPH and JPT. These data imply that the variant rs17168525 might have different effects on cardiac hypertrophy among different populations. Such a discrepancy may be caused by distinct ethnicity-related factors that affect the levels of myotrophin production.First, genetic heterogeneity of population might explain the discrepant results. Different geographical and racial backgrounds of the individuals can affect the consequences of an association study . Ethnicimyotrophin gene may exert important genetic effects on left ventricular hypertrophy risk. To the best of our knowledge, the present study is the first investigation to test the relationship between the myotrophin polymorphism and cardiac hypertrophy. Of note, gene-gene and gene-environment interactions have a larger impact on genetic susceptibility than the independent effects of each locus [Secondly, cardiac hypertrophy is known as a complex process affected by both genetic and environmental risk factors, and other variants in or near the ch locus ,32, whicmyotrophin gene may have potential functions to alter the secondary structure which in turn would determine the accessibility of the let-7/miR-98 binding at the target site.Thirdly, polymorphisms, either at the target site or around the target site of miRNA binding, might influence the secondary structure at the target site. A most recent study showed that a C-to-T polymorphism, near the miR-24 binding site on DHFR gene, can result in DHFR overexpression . In thismyotrophin variant rs17168525 may be only a part of the whole expression pool of myotrophin and the effect of human myotrophin variant rs17168525 could be diluted.Finally, another possible reason for the disparity is that human may have multiple transcripts of myotrophin. Previous report demonstrated six myotrophin transcripts in SHR heart and the levels of all the transcripts are significantly elevated in SHR hearts compared with levels of the same transcript in WKY rat hearts . Thus itin vitro study suggests that let-7/miR-98 can inhibit the expression of myotrophin, but its role in vivo remains to be further investigated. Also, the mechanisms by which the binding of let-7/miR-98 regulates cardiac hypertrophy in vivo still need to be investigated. In addition, we carried out a community-based association study between variant rs17168525 and left ventricular hypertrophy only in Han Chinese population. Further studies are needed to elucidate the role of this polymorphism in the pathogenesis of cardiac hypertrophy in various ethnic groups.There are several limitations in the present study. First, the function of let-7 family is largely unknown in heart, although a very recent study demonstrated that let-7/miR-98 negatively regulates cardiac hypertrophy . SimilarAnother limitation of the present study is that we did not have the data about the expression level of myotrophin in serum/plasma from control and hypertensive patients, thus, the association between myotrophin and development of left ventricular hypertrophy in human essential hypertension can not be well established. However, several previous studies showed that elevation of myotrophin in the plasma of patients with HF, particularly in males  and the myotrophin might increase the risk of cardiac hypertrophy by interfering with let-7/miR-98 binding. The lack of association was found between the polymorphism and left ventricular hypertrophy in a Han Chinese population. It will be important to detect mechanisms underlying the effect of this variant or other variants in or near miRNA binding target site of myotrophin and true association between myotrophin polymorphisms with cardiac hypertrophy.In summary, we report here that T allele of rs17168525 in the 3′-UTR of S1 FileA DOC file containing further tables and figures described in this article. (DOC)(DOC)Click here for additional data file.


Bacillus subtilis HSO121 are presented. Surfactins exhibit strong self-assembly ability to form sphere-like micelles and larger aggregates at very low concentrations. The amphipathic and surface properties of surfactins are related to the existence of the minor polar and major hydrophobic domains in the three 3-D conformations. In addition, the application potential of surfactin in bioremediation of oil spills and oil contaminants, and microbial enhanced oil recovery are discussed.Lipopeptides produced by microorganisms are one of the five major classes of biosurfactants known and they have received much attention from scientific and industrial communities due to their powerful interfacial and biological activities as well as environmentally friendly characteristics. Microbially produced lipopeptides are a series of chemical structural analogues of different families and, among them, 26 families covering about 90 lipopeptide compounds have been reported in the last two decades. This paper reviews the chemical structural characteristics and molecular behaviors of surfactin, one of the representative lipopeptides of the 26 families. In particular, two novel surfactin molecules isolated from cell-free cultures of  Bacillus, Agrobacterium, Streptomyces, Pseudomonas, and Thiobacillus as producers of amino acids-containing biosurfactants; Pseudomonas, Torulopsis, Candida, Mycobacterium, Micromonospora, Rhodococcus, Arthrobacter, Mycobacterium, Corynebacterium, Mycobacterium, and Arthrobacter as producers of glycolipids; Thiobacillus, Aspergillus, Candida, Corynebacterium, Micrococcus, and Acinetobacter as producers of phospholipids and fatty acids [Biosurfactants are a class of microbial metabolites with surface-active properties and they are capable of spontaneous assemblies at the air–water or water–oil interface and thereby reducing surface/interfacial tensions due to their hydrophilic and hydrophobic structural components. On the basis of their chemical structures, biosurfactants are divided into five major classes of lipopeptides, glycolipids, phospholipids, neutral lipids, and polymeric compounds . Currentty acids .Bacillus  [Pseudomonas [Streptomyces [Aspergillus [Serratia and Actinoplanes. Lipopeptides are classified as cyclic lipopeptides and linear ones based on their chemical structures [C-terminal of peptide chain bonded to the amino group from the peptide chain or a hydroxyl group from fatty acid chain. Whereas, linear lipopeptides contain a linear amino acid group connected one-by-one and the fatty acid bonded to the α-amino group or other hydroxyl group. Currently, about 90 lipopeptide compounds in 26 families have been identified and most of them were summarized previously by Liu et al. [−1 [−1 with a critical micelle concentration (CMC) of 10−5 M [As one of the typical representative biosurfactants with excellent interfacial and biological activity , lipopetolymyxa) , Pseudomudomonas , Streptoptomyces , Aspergiergillus , Serratiructures . Cyclic u et al. . The maju et al. , iturin u et al.  and fengu et al.  are most al. [−1 ,14 and tf 10−5 M .This paper presents the primary and spatial structure, property and molecular behaviors of surfactin in aqueous solution and at the interface of oil and water, and its potential application in bioremediation of oil spills and microbial enhancement of oil recovery is also discussed.et al. [et al. [l-Glu1-l-Leu2-d-Leu3-l-Val4-l-Asp5-d-Leu6-l-Leu7 [Surfactin was first reported and named in 1968 by Arima  [et al. . Surfactet al. [B. subtilis ATCC 21332 and OKB 105, two of which were different from the basic structure mentioned above in the amino acid Leu7 which was replaced by Val and Ile, respectively. Using the method reported by Yang et al. [et al. [et al. [B. subtilis when incubated with the same culture medium and found three kinds of surfactins with different peptide moieties: (1) a surfactin with amino acid sequence of N-Asp-Leu-Leu-Val-Glu-Leu-Leu-C; (2) a surfactin with amino acid sequence of N-Glu-Leu-Leu-Val-Asp-Leu-Leu-C; and (3) a surfactin with the peptide chain methyl esterified. Meanwhile, the fatty acid moiety of these surfactins was identified to be diverse, including iso C12, iso C13, anteiso C13, iso C14, n C14, iso C15, anteiso C15, n C15, anteiso C16, and anteiso C17 beta-hydroxy fatty acids. Notably, two novel surfactin molecules were isolated and identified in our laboratory in the broth of B. subtilis HSO121. One is the C12 lipopeptide [B. subtilis. The other is the C15-surfactin-O-methyl ester [B. subtilis for the first time. The fatty acid portions of C15-surfactin-O-methyl ester were identified to be iso C15 and anteiso C15 β-hydroxy fatty acids. The peptide ring was analyzed by tandem MS as shown in m/z 1072.66) and the amino acid sequence of N-Glu(OMe)-Leu-Leu-Val-Asp-Leu-Leu-C was identified by taking the differences of the fragment ions shown in 1H NMR (3.66 ppm indicating of the monoester of lipopeptide) and 13C NMR (49.15 ppm indicating the methoxy group attached on Glu or Asp). The deduced structure of C15-surfactin-O-methyl ester is shown in The differences in the sequence of amino acids and number of carbon atoms in the fatty acids means the surfactins contain a variety of structural analogues ,16,17. Bet al.  reportedg et al.  for anal [et al.  for analopeptide , which hyl ester , which iet al. [B. subtilis produced a total of 44 compounds of surfactins and their monomethyl and dimethyl esters, including the known surfactin variants with l-Leu, l-Val, or l-Ile in position 7 of the peptide ring and unknown variants with leucine residues in position 2 and/or 7 replaced by l-Val and l-Ile.Kowall et al.  also repB. subtilis which can produce surfactin, iturin and fenyins simultaneously [Different bacterial strains may produce the same surfactin compounds , and theaneously ,26,27.Much attention has been paid to the study of molecular behavior of surfactin at surface/interface because the biological activity mostly occurs at the interface and is governed by the conformational state of molecules at interfaces ,29. The 18 molecule·m−2  with surfactin concentration of 3 × 10−7 M was detected by Maget-Dana and Ptak [et al. [et al. [et al. [The characteristic of biosurfactant adsorption on the surface and/or interface has attracted more and more attention. The maximum adsorption capacity of 3 × 10and Ptak  which in [et al.  studied  [et al.  proposed [et al.  examined2, Leu3, Val4, Leu6, Leu7) and a hydrophilic moiety with the backbone of the cyclic peptide and two anionic residues (Glu1 and Asp5). This characteristic enables surfactin to tend to stay at hydrophilic/hydrophobic interfaces and consequently reduce the surface tension of aqueous solutions, which has great implications for adsorption of hydrophobic molecules, and biological assimilation and transport. From this viewpoint, the longer the fatty acid chain is in the hydrophobic moiety of surfactin, the more intensity there is in the repulsive force between water and this fatty acid chain, and hence the fast adsorption of surfactin to the surface/interface. Gang et al. [These results show that surfactin is an efficient biosurfactant. The rapid adsorption and the close alignment of surfactin on the surface or interface may be contributed to its specific amphiphilic structure with a hydrophobic moiety consisting of a long fatty acid chain and some lipophilic amino acids  and Val4 residues. Further, she found that the occurrence of hydrophobic contact between surfactin molecules increased with the interfacial concentration, especially the interactions between the hydrophobic tails. The peptide ring backbones exhibited a structural flexibility in that a more packed structure was adopted at a higher interfacial concentration  having a CMC of 9.4 × 10−6 M and γCMC (surface tension at the CMC) of 30 mN·m−1 in 0.1 M NaHCO3 solution. Liu et al. [−4 M) and ~108 nm (at 3.0 × 10−4 M) [The molecular behavior of surfactin in solution has been widely studied. Ishigami et al.  found thu et al.  studied  10−4 M) .et al. [2 to a surfactin solution. The effect of counter-ions  on micellization behavior was also studied by Li et al. [et al. [The morphology of surfactin micelle is also affected by pH, metal ions and temperature. Knoblich et al.  found thi et al.  and her  [et al.  investig2+. The micropolarity was higher at lower pH and increased in the presence of Ca2+ while the microviscosity increased with temperature, especially in the presence of Ca2+ [Not only the morphology but also the micropolarity and microviscosity of surfactin micelles are found to be affected by extrinsic factors such as pH, temperature and Ca of Ca2+ ,44.et al. [et al. [1 and Asp5 side-chains, formed a minor polar domain on one side of the ring and residue 4, lipidic chain, as well as side-chains of residues 3 and 7 constructed a major hydrophobic domain on the opposite side. In the Tsan’s structure –CO(2), fixing a β-turn) and S2 –CO(5), NH(4)–CO(2) and NH(6)–CO(carboxyl group), two of them corresponding to γ-turns) were proposed by Bonmatin et al. ,45 with  [et al.  with surtructure , the bacc domain . All the2+ ions, Vass et al. [However, based on the comparative Circular Dichroism Spectroscopy (CD) and Fourier Transform Infrared Spectroscopy (FTIR) spectroscopic studies in different solvents with or without Cas et al.  concludeThese results demonstrate that surfactin in solution possess strong self-assembly ability to form micelles and further larger aggregates and the morphology of surfactin micelles formed is notably affected by ambient conditions such as pH, metal ions and temperature.2+ complex in a molar ratio of 2:1, thus facilitating micellization via a dimer assembly, with a possible long-range effect on the spatial structure and morphology of the micelles [It is believed that the assembling ability is attributed to the spatial structure with both polar and apolar domains and the β-sheet formation of surfactin molecules ,48. The micelles . Temperamicelles .−1 with a CMC of 10−5 M and is the most effective biosurfactant in terms of fundamental dynamic and equilibrium interfacial properties [Surfactin exhibits a strong surface activity by reducing the surface tension of water to 27 mN·moperties . In addioperties .l-Gln1-d-Leu2-l-Leu3-l-Val4-l-Asp5-d-Leu6-l-Ile7-β-OH fatty acid). Surface tension of lichenysin A solution were found to decrease with the increase in the percentage of branched chain fatty acids and increase when the percentage of straight chain 3-hydroxy tetradecanoate increased [et al. [The differences in structure, the carbon length and structure of fatty acid, amino acid composition as well as their sequence in the peptide chain, have some specific impact on the property of surfactins. Generally, surfactins showed a stronger ability in reducing both the dynamic and equilibrium surface tensions with more hydrophobic alkyl chains ,33,51 anncreased . With reet al. [et al. [Linear surfactin shows lower surface activities and poor hemolytic activities compared to those of cyclic structures . Eeman eet al.  found th [et al.  demonstr [et al. . These ret al. [O-methyl ester (O-methyl ester had a stronger surface activity than C15-surfactin. C15-surfactin-O-methyl ester showed CMC of 9.78 × 10−6 M and surface tension at CMC (γCMC) of 28.71 mN/m in 10 mM phosphate buffered saline , whereas, C15-surfactin was characterized with CMC of 1.74 × 10−5 M and γCMC of 30.03 mN/m under the same conditions. Morikawa et al. [et al. [et al. [B. licheniformis with the surfactin produced by B. subtilis  and found that lichenysin was with a higher surfactant power  than surfactin .The two Glu and Asp residues present in the cycle of surfactins play a critical role in their amphiphilic properties. Liu et al.  studied yl ester  with coma et al.  found th [et al.  studied  [et al. ,57. Gran [et al.  conducte4 by a more hydrophobic residue, i.e., leucine or isoleucine and found that these Leu4- and Ile4-surfactins had a higher affinity for hydrophobic solvents and a twofold decrease of CMC [In terms of the role of amino acids in the modulations of activities, Bonmatin studied the properties of surfactin modifiers with the substitution of the L-valinee of CMC . In lighe of CMC ,59.The ambient pH also affects the surface/interface property of surfactin markedly. Surfactins show higher activity under alkaline conditions than under acidic conditions. When aspartic acid residues and glutamic acid of surfactins were amido-esterified or methyl-esterified, the obtained derivatives retained similar activities irrespective of the pH changes .l-Glu1-l-Leu2-d-Leu3-l-Val4-l-Asp5-d-Leu6-l-Leu7), obviously presents the amphiphilic character. Based on all experimental data mentioned above, the three-dimensional structures proposed by Bonmatin et al. [et al. [Surfactin, with β-hydroxy fatty acids linked to a heptapeptide  were positive in oil production [4 t additional crude oil over 10 years [In the past decades, about 10 MEOR applications have been implemented in Malaysia, USA, Argentina, and China and a successful MEOR field test was observed with oil reservoir temperatures below 55 °C, salinity less than 100,000 ppm, water cut above 75%, and a low production rate . Maudgaloduction . Till no10 years . With alOil spills are one of the major causes of oil pollution in marine environments. Due to the toxic and persistent characteristics, chemically synthesized surfactants are not favored as remediating agents.et al. [Biosurfactant is a potential alternative for bioremediation of crude oil contamination and oil spills in marine environments and other terrestrial ecosystems for its excellent surface activity and emulsifying properties as well as lower CMC characteristics. It has been shown that from the above CMC, stable aggregates are formed  which can increase the solubility of hydrophobic organic contaminants, providing opportunities for phyto- or microbial remediation. The ability of biosurfactants in emulsifying hydrocarbons into water to form mixtures has been widely reported . These eet al.  isolatedet al. . Meanwhiet al.  as well et al. . These fin situ in the contaminated environment will be of great significance in the oil clean up. Laboratory studies have shown that the addition of biosurfactant mixtures alone is useful for stimulating biodegradation of hydrocarbon contaminants in the environment [in situ bioremediation of oil contaminated soil by biosurfactant-producing B. subtilis was also demonstrated [Biosurfactants produced by one consortium of microorganisms may enhance the biodegradation of oil hydrocarbons by other microorganisms. Considering the ubiquitous existence of marine hydrocarbon-degrading bacteria—the obligate hydrocarbonoclastic bacteria (OHCB) which have been recognized and shown to play a significant role in the biological removal of petroleum hydrocarbons from polluted marine waters ,85,86—biironment . The widironment . The effnstrated .Generally, oil-degrading microorganisms have the capacity to produce biosurfactants to facilitate their growth by enhancing assimilation of hydrocarbons and nutrients available from the environment. Even in the cold oceanic environment, some oil-degrading strains can thrive and degrade oil normally and the low temperature had no obvious effects on the hydrocarbon degrading bacteria community and the bioremediation process . A numbeBiosurfactants were experimentally investigated for their ability in facilitating the removal of adherent oil from Alaskan beaches and results demonstrate that microbial surfactants released oil to a significantly greater extent (two to three times) than water alone . The abiet al. [The results of field experiments and trials following actual spill incidents have been reviewed by Swannell et al. . The oilEvaluation methods have been established . DispersB. subtilis RB14 was shown to be effective for the control of damping-off caused by R. solani in tomato plants [B. subtilis 20B showed antifungal activity against mycelia growth of C. indicum, A. burnsii, F. oxysporium, F. udum, T. herzanium and R. bataticola [B. subtilis have the most potent antibiotic activity [B. licheniformis inhibits germination of M. grisea [et al. [B. subtilis possessed a broad spectrum of activity against Gram-negative bacteria, little activity against Gram-positive organisms, as well as one fungi assayed.Being amphiphilic membrane active biosurfactants, surfactin, fengycin and iturin compounds are found to be of potent antimicrobial activities. Iturin are potent antifungal agents which can be used as biopesticides for plant protection. Iturin A produced by o plants . B. subttaticola . Surfact. grisea . Bechard [et al.  demonstret al. [Taking into consideration the amphiphilic nature of these lipopeptides, the above-mentioned activities are presumably believed to be a direct consequence of the interaction of lipopeptide with its target membrane and the alteration of the bilayer properties. The interaction of lipopeptides with membrane lipids may form a pore which causes cellular leakage ,99. Carret al.  observedet al. .Also, biosurfactants have found application or prospect in cosmetics , the fooBiosurfactants are a unique class of microbial metabolites with surface-active properties. The differences in the sequence of amino acids and carbon atoms in the fatty acids provide surfactins with diverse chemical structures and physiochemical properties. The amphiphilic characteristics of surfactin molecules enable them to have a strong tendency to adsorb to hydrophilic/hydrophobic interfaces and, consequently, reduce the surface tension of aqueous solutions, leading to the formation of a stable insoluble monolayer in the gas–liquid interface. The physical states of this monolayer are influenced by subphase temperature and pH. The fact that surfactins exhibit a strong self-assembly ability to form micelles and some larger aggregates, even at rarely low concentrations, can be reasonably explained by these three typical 3-D structures that are developed. The amphiphilic and surface properties of surfactins are certainly related to the existence of the minor hydrophilic and major hydrophobic domains as described in the three 3-D conformations. Recently, due to several advantages over chemical surfactants such as lower toxicity, high environmental compatibility, biodegradability, and synthesis from renewable raw materials, biosurfactants have been the focus of extensive research and applications, especially in microbial enhanced oil recovery as well as remediation of oil spills.


A prototype 1024 × 32 Time Delay Integration (TDI) CMOS image sensor with the proposed ADC architecture has been fabricated in a standard 0.18 μm CMOS process. The proposed ADC has average power consumption of 128 μW and a conventional rate 6 times higher than the conventional SS ADC. A high-quality image, captured at the line rate of 15.5 k lines/s, shows that the proposed ADC is suitable for high-speed CMOS image sensors.A 12-bit high-speed column-parallel two-step single-slope (SS) analog-to-digital converter (ADC) for CMOS image sensors is proposed. The proposed ADC employs a single ramp voltage and multiple reference voltages, and the conversion is divided into coarse phase and fine phase to improve the conversion rate. An error calibration scheme is proposed to correct errors caused by offsets among the reference voltages. The digital-to-analog converter (DAC) used for the ramp generator is based on the split-capacitor array with an attenuation capacitor. Analysis of the DAC's linearity performance  Although high-speed SS ADCs have been recently reported  to , which can be expressed as:On the other hand, the maximum DNL is expected to occur when the binary code changes from , then the 8 bits, [b9b8…b2], were chose to obtain 8. Then [b7b6…b0] were chose to obtain When measuring under a relatively low illumination condition, all the digital outputs ranged from 0 to 2versus illumination and measured INL for an output range between 20% and 90% of saturation. The non-linearity comes from the pixel, the analog accumulator and the column-parallel ADC. versus illumination. In addition, the measured ADC column FPN is 0.15%, which is close to the conventional SS ADC. The performance of the proposed imager is listed in The non-linearity of the CIS is measured according to the method in . Figure 5.A 12-bit high-speed column-parallel two-step SS ADC for CMOS image sensors is proposed. The proposed ADC employs a single ramp voltage and multiple reference voltages, and the conversion is divided into coarse phase and fine phase to improve the conversion rate. An error calibration scheme is proposed to correct error caused by offsets among the reference voltages. A prototype 1024 × 32 TDI CMOS image sensor with the proposed ADC architecture has been fabricated in a standard 0.18 μm CMOS process. The proposed ADC has average power consumption of 128 μW and conversion time of 36 μs. The proposed ADC is much faster than the conventional SS ADC, and costs less power and area compared with the multiple-ramp ADC. Simulation results indicate that the SNDR and ENOB with the proposed calibration are 72.02 dB and 11.67-bit, respectively. The measured FPN is 0.15%. A high-quality image, captured at the line rate of 15.5 k lines/s, shows that the proposed ADC is suitable for high-speed CISs.


Coprinopsis cinerea contains a quinohemoprotein (CcPDH named as CcSDH in our previous paper), which is a new type of pyrroloquinoline-quinone (PQQ)-dependent pyranose dehydrogenase and is the first found among all eukaryotes. This enzyme has a three-domain structure consisting of an N-terminal heme b containing a cytochrome domain that is homologous to the cytochrome domain of cellobiose dehydrogenase  from the wood-rotting basidiomycete Phanerochaete chrysosporium, a C-terminal family 1-type carbohydrate-binding module, and a novel central catalytic domain containing PQQ as a cofactor. Here, we describe the biochemical and electrochemical characterization of recombinant CcPDH. UV-vis and resonance Raman spectroscopic studies clearly reveal characteristics of a 6-coordinated low-spin heme b in both the ferric and ferrous states, as well as intramolecular electron transfer from the PQQ to heme b. Moreover, the formal potential of the heme was evaluated to be 130 mV vs. NHE by cyclic voltammetry. These results indicate that the cytochrome domain of CcPDH possesses similar biophysical properties to that in CDH. A comparison of the conformations of monosaccharides as substrates and the associated catalytic efficiency (kcat/Km) of CcPDH indicates that the enzyme prefers monosaccharides with equatorial C-2, C-3 hydroxyl groups and an axial C-4 hydroxyl group in the 1C4 chair conformation. Furthermore, a binding study shows a high binding affinity of CcPDH for cellulose, suggesting that CcPDH function is related to the enzymatic degradation of plant cell wall.The basidiomycete  CcPDH named as CcSDH in our previous paper) that represents a new type of pyrroloquinoline quinone (PQQ) quinohemoprotein from the basidiomycete Coprinopsis cinerea  strain 5338. This CcPDH is the first such protein found among all eukaryotes [CcPDH indicates that the extracellular enzyme contains three domains: an N-terminal cytochrome domain, a C-terminal carbohydrate binding module (CBM), and a central PQQ domain.Cellulolytic fungi are known to produce many extracellular oxidoreductases, such as peroxidase and laccases, as well as cellulolytic enzymes –4. RecenCcPDH has 32 to 42% identity with the cytochrome b domains of basidiomycete cellobiose dehydrogenases (CDHs). CDH is a major oxidoreductase secreted by many cellulolytic fungi during growth on cellulose, and the genes that code for CDHs have been identified in numerous cellulolytic fungi [b containing a cytochrome domain, and a linker region connecting the two domains. CDH catalyzes the dehydrogenation of cellobiose and cello-oligosaccharides to their corresponding δ-lactones. The oxidation of cellobiose takes place in the FAD domain, subsequently followed by inter-domain electron transfer to the cytochrome domain [b-type heme with unusual Met-His ligands [www.cazy.org), the FAD and cytochrome domains of CDH are classified in subfamily 1 of Auxiliary Activity family 3 (AA3_1) and AA8, respectively [CcPDH has an AA8 cytochrome domain but a cellulose-binding domain attached to a PQQ domain instead of an FAD domain.The N-terminal cytochrome domain of ic fungi . CDH is e domain . The cyt ligands . Because ligands , 10. Accectively . CcPDH hc-type heme cofactors [Acinetobacter calcoaceticus [Escherichia coli [Pyrobaculum aerophilum [A. calcoaceticus sGDH. These Asds have oxidation activity towards a broad range of aldose sugars, with a weak affinity for substrates. The central PQQ domain of CcPDH has low identity with sGDH from A. calcoaceticus (16%). The putative homologues of the PQQ domain of CcPDH are widely distributed in bacteria, archaea, amoebozoa, and fungi. These quinoproteins belong to a different phylogenetic category from the known PQQ-quinoprotein family [CcPDH is currently identified as a new Auxiliary Activities family 12 (AA12) in the CAZy database.Several known prokaryotic dehydrogenases contain PQQ as their prosthetic group and mainly catalyze the oxidation of a number of alcohols or aldose sugars in the periplasm of Gram-negative bacteria –14. In tofactors , 18. Theofactors , 20 and aceticus , 22. In hia coli  and Pyrorophilum  are PQQ n family . TherefoCcPDH indicates the presence of a family 1 CBM (CBM1). The majority of cellulose-binding domains attached to fungal cellulolytic enzymes belong to CBM1. The main role of CBM1 in cellulolytic enzymes is to enhance the hydrolysis of crystalline cellulose by increasing the effective enzyme concentration on the cellulose surface [Many carbohydrate active enzymes including hydrolytic and non-hydrolytic proteins contain one or more non-catalytic CBMs that have functions in recognizing and adhering to carbohydrates –27. Base surface , 30. CBM surface –35. The  surface .CcPDH is an attractive multi-domain oxidoreductase that has three unique domains with different backgrounds. Until now, CcPDH had been overexpressed in the methylotrophic yeast Pichia pastoris, and initial characterization of the recombinant protein has demonstrated the PQQ-dependence of the enzyme reactivity for the first time in a eukaryotic enzyme. Despite its low amino acid sequence homology with known PQQ-binding enzymes, this enzyme binds PQQ strongly and shows oxidation activity towards some sugars. In this study, we thoroughly investigated the enzymatic and electrochemical properties of recombinant CcPDH.As noted above, c were purchased from Sigma-Aldrich Japan G. K. . 2-Keto-D-glucose was purchased from Santa Cruz Biotechnology, Inc. . l-fucose, l-lyxose, and l-xylose were purchased from Wako Pure Chemical Industries . d-arabinose and l-mannose were purchased from Sigma-Aldrich Japan G. K. . l-galactose, d-talose, and l-rhamnose were purchased from Funakoshi Co., Ltd. . l-gulose and l-glucose were purchased from Tokyo Chemical Industry Co., Ltd. .Pyrroloquinoline quinone and equine heart cytochrome CcPDH and PQQ containing dehydrogenase domain (DHPDH) of CcPDH (DHPDH: residues 240–649) were heterologously expressed in the methylotrophic yeast Pichia pastoris and purified as described previously [CcPDH” in this paper means the heterologously expressed wild type. The purity of CcPDH was confirmed by SDS/PAGE and by absorption spectroscopy. The CcPDH concentration in the solution was estimated from the absorbance at 420 nm (ε420 = 130 mM-1 cm-1) [Recombinant intact eviously . The enz-1 cm-1) .CcPDH and DHPDH were recorded at room temperature with a JASCO V-660 spectrophotometer . CcPDH was diluted in 50 mM HEPES buffer, pH 7.0, containing 1 μM PQQ and 1 mM CaCl2. Ca2+ is ligated with PQQ and several amino acid side chain atoms and is essential for the formation of active holo-enzyme of quinoproteins. The reduced form of CcPDH was prepared by addition of 1 mM ascorbic acid or l-fucose. Apo-DHPDH was stored in 50 mM HEPES buffer, pH 7.0, containing 1 mM CaCl2. Holo-DHPDH was obtained by addition of PQQ to apo-DHPDH according to our previous report [PDH was prepared by addition of 1 mM l-fucose.Electronic absorption spectra of s report . The red2-cooled CCD detector . Raman scattering was excited by the 413.1 nm emission from a Kr ion laser , and the incident powers were 1 mW. The spectra were obtained with data accumulated over 30 sec and a spectral resolution of 1.47 cm-1. The spectra were calibrated against indene and CCl4 as external standards. A two-point baseline was performed with GRAMS/386 software. All measurements were carried out in 50 mM HEPES buffer (pH 7.0) containing 1 μM PQQ and 1 mM CaCl2, at room temperature. The reduced form of CcPDH was prepared by the addition of 1 mM ascorbic acid or l-fucose.Resonance Raman (RR) spectra were measured on a JASCO NRS-1000 spectrometer using a Kaiser Optical holographic notch-plus filter and a liquid NCcPDH were analyzed by cyclic voltammetry . The potential was determined by averaging the anodic and cathodic peak potentials. The electrochemical responses of CcPDH were obtained by using a carbon nanoparticle-modified plastic-formed carbon as a working electrode, as described elsewhere [2 using a three-electrode cell under a nitrogen atmosphere at room temperature.Redox responses of heme in lsewhere . An Ag/ACcPDH were assayed using cytochrome c as an electron acceptor [c at 550 nm (Δε550 = 17.5 mM-1 cm-1) at 30°C. To obtain the kinetic parameters of oxidation for l-galactose, d-talose, l-xylose, and l-glucose, the assay was performed with various concentrations of monosaccharides  in 50 mM Tris buffer, pH 8.5 containing 1 μM PQQ and 1 mM CaCl2. To determine the Michaelis constant (Km) and turnover number (kcat), experimental data were fitted with the Michaelis-Menten equation with using Origin 8.1  The activity of DHPDH was determined using a dye-linked assay with the artificial electron acceptors phenazine methosulfate (PMS) as a primary electron acceptor and 2,6-dichlorophenolindophenol (DCPIP), according to our previous report [PDH toward l-fucose was measured in 50 mM MES buffer, pH 6.5, containing 0.1 mM DCPIP, 1 mM PMS, 1 mM CaCl2, 100 mM l-fucose, 50 nM DHPDH, and varying concentrations of PQQ (2.5–100 nM). The reaction rate was measured by following the decrease in the absorbance of DCPIP at 520 nm (Δε520 = 6.9 mM-1 cm-1) [The enzyme activities of acceptor , 39, 40.s report . The eff-1 cm-1)  at 30°C CcPDH sequence were subjected to multiple-alignment analysis using MAFFT (ver. 6.85) [The amino acid sequences of CBM1s within carbohydrate-active enzymes were collected from the CAZy database. The CBM1 sequences and the r. 6.85) , 43, andr. 6.85) .Cladophora sp. and phosphoric-acid-swollen cellulose (PASC) prepared from Avicel according to the method described previously [CcPDH (0.5–32 μM) were incubated with 0.1% w/v celluloses for 1 h at 30°C and then were centrifuged for 20 min at 16,100 g. The absorbance at 420 nm, which corresponded to the Soret band of a b-type heme (ε420 = 130 mM-1 cm-1), was measured with UV-vis spectroscopy to determine the concentration of free enzyme in the supernatants. The equilibrium dissociation constant (Kd) and the maximum amount of adsorbed enzyme (Amax) were estimated as described previously [Adsorption experiments were carried out in 50 mM HEPES buffer, pH 7.0, using crystalline cellulose from eviously  as an ameviously , 47.CcPDH are shown in CcPDH by ascorbic acid, sodium dithionite, or l-fucose, which is a substrate, induced the same behavior, and no difference was observed in the resulting spectra. The absorbances of the γ-, β-, and α-bands were increased and the maxima of the bands shifted slightly to 429 nm, 532 nm, and 562 nm, respectively. The spectra shifts due to the reaction with l-fucose suggests that the electron generated by l-fucose can be transferred from the PQQ domain to the cytochrome domain, like that from the FAD domain to the cytochrome domain in the cellobiose oxidation of CDH.The absorption spectra of the oxidized CcPDH, RR spectra were measured for both the oxidized and reduced forms of CcPDH with excitation at 413 nm as shown in 4 of CcPDH in its oxidized form appears at 1370 cm-1, indicating a ferric heme. The core size marker bonds ν2 and ν3 of the oxidized form, occurring at 1574 and 1502 cm-1, respectively, identifies the ferric heme as having a hexacoordinate low-spin (6cls) heme state. The reduced form of CcPDH prepared with either ascorbic acid or l-fucose showed the same RR spectral bands with the ν2, ν3, and ν4 to 1577, 1492, and 1360 cm-1, respectively. These values indicate that the reduced CcPDHs have a ferrous heme with a 6cls state. The RR spectral data of the oxidized and the reduced forms of CcPDH are summarized in P. chrysosporium. The RR frequencies of oxidized and reduced CcPDH were near agreement with those of CDH, suggesting that these cytochrome proteins have a similar microenvironment around the heme cofactor.Both the oxidized and reduced heme cofactors are RR active chromophores, and the vibrational modes can be enhanced with laser excitation in the 400–500 nm range. To further investigate the properties of the heme in CcPDH was estimated by cyclic voltammetry. As shown in CcPDH and the electrode. The redox potential determined from the midpoint of the oxidation-reduction peak potentials is 130 mV vs. NHE at pH 7. This is in accordance with previous reports describing the cytochrome domain of CDH from P. chrysosporium in solution  [3+/Fe2+ redox couple of the heme b center in CcPDH.The reduction-oxidation (redox) potential of V, pH 7) . Thus, tCcPDH, we used DHPDH that was separated from a cytochrome domain and a CBM1 by a linker region. The UV-visible spectrum of the purified DHPDH was typical of an enzyme without a cofactor. It had an absorbance maximum at 279 nm with aromatic amino acid residues  of 0.99. This value increases in linear manner to the equivalence point of the PQQ, indicating that the affinity of apo-DHPDH for PQQ is high, similar to bacterial PQQ quinoproteins [PDH binds PQQ with a 1:1 stoichiometry and has strong affinity with a dissociation constant (Kd) of 1.11 nM [CcPDH is a PQQ-dependent dehydrogenase.proteins , 49, 50.CcPDH catalyzes the dehydrogenation of monosaccharides having a 1C4 chair conformation, such as l-fucose and d-arabinose [l-galactose, d-talose, l-xylose, l-glucose, l-lyxose, l-mannose, and l-rhamnose (= 6-deoxy- L-mannose), noting the configuration of the substrate as shown in kcat value towards l-galactose was comparable to l-fucose, which is a 6-deoxy sugar of l-galactose, whereas the Km values of l-galactose were twice that of l-fucose. The affinity for d-talose was about the same as for l-fucose, but the kcat value was decreased by 30%. The Km values for l-xylose and l-glucose were approximately 236 mM and 363 mM, respectively, which are markedly greater than the Km obtained for the former monosaccharides. Slight oxidation activities of CcPDH were observed for l-lyxose and l-mannose, but little was observed for l-rhamnose. Thus, the catalytic efficiency was highest for d-glucosone, followed by l-fucose > d-arabinose ≈ l-galactose > d-talose ≈ l-gulose > d-lyxose > l-xylose ≈ l-glucose.Our previous studies suggest that rabinose . We examCcPDH indicated the presence of a CBM Family 1 C-terminal domain of CcPDH. The CBM1 homologous sequence of CcPDH was aligned with other CBM1s from fungal cellobiohydrolases (CBHs), ß-glucosidase (BGL), carbohydrate-binding cytochrome b562 (CBCyt. b562) and the basidiomycete CDH using MAFFT multiple alignment, as shown in CcPDH contains three conserved aromatic residues  that are proposed to contribute to CcPDH binding on the surface of carbohydrates. It also shows conserved cysteines  involved in disulfide bond formation. The amount of CcPDH adsorbed on insoluble cellulose was measured at various protein concentrations after incubation for 2 hours. A highly crystalline cellulose Ia from Cladophora and a PASC, which is an amorphous cellulose obtained by phosphoric acid treatment, were used as carbohydrate materials. Amax and Kd from the corresponding plots are shown in Amax on PASC showed higher adsorption by CcPDH than on cellulose Ia from Cladophora and the binding efficiency (Amax/Kd) for PASC was approximately 11 times higher than that for cellulose Ia. This is likely due to the difference in the surface area where the enzyme can adsorb. Therefore, the results suggest CcPDH is a redox enzyme that has the ability to adsorb on cellulose surfaces as well as CDHs.The amino acid sequence of P. chrysosporium, CcPDH was discovered in the C. cinerea 5338 strain as a new quinohemoprotein. CcPDH has a three-domain organization, with a catalytic domain containing PQQ as a cofactor (DHPDH), a b-type cytochrome domain as a redox site, and a CBM1. As far as we know, this is the first example of an extracellular quinohemoprotein connected to a cytochrome domain of CDH and CBM1. In this study, the spectroscopic and electrochemical properties, substrate specificity, and carbohydrate-binding properties of this novel PQQ-dependent enzyme from C. cinerea were determined.In a previous homology search with the cytochrome domain of CDH from CcPDH shows significant homology to that of CDH, with well-conserved Met/His ligands for heme binding and a disulfide bond. The structural homology modeling of the cytochrome domain of CcPDH by the Phyre program provided structural folds into an immunoglobulin-like ß-sandwich consisting of a five-stranded and a six-stranded ß-sheet with a short α-helical structure at the C-terminus, which is an unusual module among cytochromes [CcPDH in the oxidized and reduced forms are in good agreement with those previously reported for the cytochrome domain of CDH from P. chrysosporium. Those studies confirm a 6cls heme b in both the ferric and ferrous states with Met/His ligands, like CDH. In addition to spectroscopic results, electrochemical studies indicate that the redox potential of the heme is +130 mV, which is comparable to CDH from P. chrysosporium. These observations suggest that the cytochrome domain of CcPDH may have a similar electron transfer function to the cytochrome domain of CDH, i.e., intra- and inter-molecular electron transfer between the cytochrome domain and the catalytic domain and also Fe (III)-reducing ability. Indeed, the electron transfer between CcPDH and cytochrome c can be observed, as it is commonly used as an electron acceptor in the CDH reaction. CDHs are well known as redox enzymes capable of DET between the enzyme and the electrode and the cytochrome domain can act as a built-in electron-transfer mediator [CcPDH was also capable of DET. Thus, CcPDH is an excellent candidate for construction of DET-system bioelectronic devices such as biosensors and biofuel cell anodes, as well as CDHs.The N-terminal cytochrome domain of ochromes . The elemediator , 10, 51.CcPDH exhibited dehydrogenase activity towards monosaccharides in a 1C4 chair conformation, in contrast to the substrate specificity of A. calcoaceticus sGDH, which has an absolute preference for l-arabinose and d-xylose in the 4C1 conformation over the d-arabinose and l-xylose enantiomers [E. coli also lacks activity for l-hexoses or pentoses that have a 1C4 chair conformation, such as l-glucose, l-mannose, l-rhamnose, and l-xylose and d-pentoses [E. coli exhibited no preference for different enantiomers with d- and l-arabinose [Interestingly, ntiomers . The mempentoses . Additiorabinose .CcPDH for these substrates and the side group orientation of each monosaccharide when in a 1C4 conformation are summarized in d-glucosone and l-fucose in d-glucosone (= 2-keto-D-glucose) has four tautomer forms reported by Freimund et al [1C4 conformation of 2-keto-D-glucopyranose is the one of the tautomer, at 21% abundance in aqueous solution, as shown by d-glucosone are shown with the corresponding carbon number of the other monosaccharides in a 1C4 conformation . The Km value for l-gulose, which is a C-3 epimer of l-galactose, was approximately 1.7-fold higher than that of l-galactose. This value indicates that the C-3 hydroxyl group in the equatorial conformation is preferred over the axial for CcPDH. The affinity for pentose sugars, which lack a hydroxymethyl group at the C-5 position, or a 6-deoxy-hexose, which has methyl group at C-5 (in L-fucose), increased over hexose sugars that have a C-5 hydroxymethyl group, suggesting that the hydroxymethyl group at this position causes some steric hindrance. However, the result suggests that C-5 methyl group is not critical for binding to CcPDH, as indicated by the oxidation of l-fucose. In light of the stereospecificity of other PQQ-dependent dehydrogenases [CcPDH may catalyze the dehydrogenation reaction of regioselective substrates at the C-1 hydroxyl group, although we can not rule out side reactions at other positions. Further studies are necessary to identify a product of CcPDH oxidation and they are currently under consideration.The compared catalytic activity of nd et al . The 1C4ormation . The reslucosone . The equogenases , 52, 55,CcPDH shows high affinity toward microcrystalline and amorphous cellulose. This result suggests the possibility of localizing CcPDH on the surface of cellulose, as is the case for CDHs. Further cytochemical analysis will be needed to identify the localization of CcPDH in nature.Many extracellular fungal carbohydrate hydrolytic enzymes carry a CBM1, which is thought to facilitate binding to the surface of cellulose chains and is related to the catalytic function of these enzymes. Although these CBM1 are not found in basidiomycetous CDHs, the flavocytochrome enzymes are also capable of binding to cellulose by its flavin domain and localizes on cellulose surfaces –58. In tCcPDH could allow direct electron transfer for LPMO as well as that of CDH. However the physiological functions remain obscure in vivo. It is not only some redox proteins instead of CDH, but some reductive compounds are the possibly electron donor for LPMO in those fungi. Indeed, l-ascorbic acid is typically used as an electron donor for LPMO in vitro [d-glucosone by CcPDH could be an intermediate in the l-ascorbic acid biosynthesis [l-ascorbic acid [Penicillium cyaneo-fulvum has an extracellular d-erythorbic acid (isoascorbic acid) synthesis pathway by using glucose oxidase and gluconolactone oxidase [CcPDH, therefore, may play a role in the l-ascorbic acid biosynthesis pathway and then LPMOs could be provided with electrons from these reductive compounds. Anyhow, CcPDH must be related to the enzymatic degradation of the plant cell wall because of its capability of binding to cellulose.Under aerobic conditions, cellulose degradation by cell-free culture filtrates of various fungi has shown much higher rates compared to anaerobic conditions, suggesting that an oxidative reaction may play a crucial role in the fungal degradation of cellulose. CDH is the first example of an oxidoreductase related to the extracellular oxidative process for cellulose degradation. Furthermore, it has been recently reported that the cytochrome domain of CDH can transfer electrons to fungal copper-dependent lytic polysaccharide monooxygenases (LPMOs) that were classified in AA9. The combination of CDHs and LPMOs synergistically enhances cellulase activity –62. The in vitro . The oxiynthesis . PQQ quibic acid , 65, 66. oxidase . CcPDH, CcPDH, which was classified as a member of the new AA12 family in CAZy using spectroscopic and electrochemical methods. These enzymatic properties provide new insight into other known PQQ quinoproteins as well as the basidiomycetous oxidoreductase network. The N-terminal cytochrome domain of CcPDH contains a 6-coordinated low-spin heme b in both the ferric and ferrous states with Met/His ligands as well as CDH and it enables DET between CcPDH and the electrode. The catalytic domain shows oxidation activity toward monosaccharides in a 1C4 chair conformation in the presence of PQQ as a cofactor, and the most efficient catalysis was observed for d-glucosone. CcPDH exhibited binding affinity for insoluble cellulose. These results suggest that CcPDH may be involved in the extracellular oxidative degradation as well as CDH. Therefore, these proteins are an attractive component of cellulolytic enzymes for use in biodegradation and biomass conversion or as an anode catalyst for bioelectrochemical applications.We have demonstrated the full characterization of


The presence of impaired glucose tolerance (IGT) and metabolic syndrome (MetS) are two risk factors for Type 2 diabetes. The inter-relatedness of these factors among adolescents is unclear.We evaluated the sensitivity and specificity of MetS for identifying IGT in an unselected group of adolescents undergoing oral glucose tolerance tests (OGTT) in the National Health and Nutrition Evaluation Survey 1999–2010. We characterized IGT as a 2-hour glucose ≥140 mg/dL and MetS using ATP-III-based criteria and a continuous sex- and race/ethnicity-specific MetS Z-score at cut-offs of +1.0 and +0.75 standard deviations (SD) above the mean.Among 1513 adolescents, IGT was present in 4.8%, while ATP-III-MetS was present in 7.9%. MetS performed poorly in identifying adolescents with IGT with a sensitivity/specificity of 23.7%/92.9% for ATP-III-MetS, 23.6%/90.8% for the MetS Z-score at +1.0 SD and 35.8%/85.0 for the MetS Z-score at +0.75 SD. Sensitivity was higher (and specificity lower) but was still overall poor among overweight/obese adolescents: 44.7%/83.0% for ATP-III-MetS, 43.1%/77.1% for the MetS Z-score at +1.0 SD and 64.3%/64.3% for MetS Z-score at +0.75 SD.This lack of overlap between MetS and IGT may indicate that assessment of MetS is not likely to be a good indicator of which adolescents to screen using OGTT. These data further underscore the importance of other potential contributors to IGT, including Type 1 diabetes and genetic causes of poor beta-cell function. Practitioners should keep these potential causes of IGT in mind, even when evaluating obese adolescents with IGT. The persistent high prevalence of pediatric obesity has greatly increased risk of Type 2 diabetes mellitus (T2DM) in the current generation of children and adolescents, increasing the need for effective tools to predict those at highest risk ,2. One sAnother important potential tool to screen for risk of T2DM is a set of criteria to identify the metabolic syndrome (MetS), a cluster of cardiovascular risk factors that occur together more often than would be expected by chance . These fIt is unknown what the short-term risk of progression to T2DM is among adolescents with MetS; however, MetS appears to be a less specific marker than IGT of imminent risk of T2DM, given that MetS is classified in 8.6% percent of adolescents in the US  while thOur goal was to evaluate the potential for MetS to identify individuals with IGT, as an important precursor to T2DM. We utilized data from the National Health and Nutrition Evaluation Survey (NHANES) data with two hypotheses: 1) that traditional MetS would have a high sensitivity and specificity for predicting IGT and 2) that our sex- and race/ethnicity-specific risk score would exhibit improved sensitivity to identify adolescents with IGT. In doing so we hoped to clarify relationships between these markers of long-term risk T2DM risk among adolescents.http://www.cdc.gov/nchs/nhanes.htm). The NCHS ethics review board reviewed and approved the survey and participants gave informed consent prior to participation.Data were obtained from NHANES (1999–2010), a complex, multistage probability sample of the US population. These annual cross-sectional surveys are conducted by the National Center for Health Statistics (NCHS) of the Centers for Disease Control (CDC), with randomly-selected participants undergoing anthropometric and blood pressure measurements, answering questionnaires and undergoing phlebotomy , and laboratory measures of triglycerides, HDL-C, and glucose were obtained using standardized protocols and calibrated equipment . All bloA fasting oral glucose tolerance test was performed on a random sub-set of adolescent participants of NHANES. After their fasting glucose assessment, participants were given 1.75 g/kg of glucola to a maximum of 75 g. Two hours after this ingestion a second blood draw was obtained to assess 2-hour glucose level.Data from non-Hispanic-white, non-Hispanic-black, or Hispanic (Mexican-American/other Hispanic) adolescents 12-19 y were analyzed. Children <12 y were excluded since fasting values for triglycerides and glucose were only obtained in participants ≥12 y. Participants with known diabetes (T1DM or T2DM) were excluded by eliminating those with self-reported diabetes and those on anti-diabetic medications. Participants were also excluded if they were pregnant or taking antihyperlipidemic medications as these are likely to alter lipid and glucose levels in a manner that may reflect baseline relationships between MetS and glucose tolerance. Individuals taking anti-hypertensive medication were classified as having hypertension.http://publichealth.hsc.wvu.edu/biostatistics/mets).IGT was classified for a two-hour blood glucose of ≥140 mg/dL. MetS status was evaluated in two manners: 1) A commonly-used pediatric/adolescent adaptation of the Adult Treatment Panel III (ATP III) criteria ,29. Partth percentile among lean adolescents in NHANES [Statistical significance was defined as a p-value < 0.05. Statistical analysis was performed using SAS . Prevalence rates of MetS were calculated by glucose tolerance category, and compared via chi-square tests. The presence of insulin resistance was categorized based on an elevated fasting insulin ≥16.0 IU/mL, approximately the 95n NHANES  and usedn NHANES -34. Receth percentile among lead adolescents, 51.9 vs. 20.1, p < 0.0001). HbA1c values were similar between groups, as was the MetS Z-score.We analyzed a study sample consisting of 1513 non-Hispanic-white, non-Hispanic-black and Hispanic adolescents age 12-19 y with data for all variables tested. Overall 4.8% of participants exhibited IGT while 7.92% were classified as having MetS  had a sensitivity of 23.6% and specificity of 90.8%  but an improved sensitivity among non-Hispanic blacks (55.9% vs. 31.8%). This improvement in sensitivity among non-Hispanic black adolescents may not be surprising, since traditional MetS criteria utilize population-based cut-off values for the individual components and do not take into account that non-Hispanic-black adolescents have lower baseline levels of triglycerides and are less likely to exhibit abnormalities in triglycerides or HDL despite having more insulin resistance and higher rates of diabetes -26. ThesHowever, while we noted an increased sensitivity using a lower cur-off of the race/ethnicity-specific MetS Z-score, the presence of MetS overall was a poor screening test to identify adolescents with IGT. Acceptable degrees of sensitivity and specificity in a screening test depend on the importance of the outcome being screened for and the downside to missing detection of that outcome. In this case, the specificity of these tests was overall reasonable clinically, 78-97% among all adolescents. However, the low sensitivity and thus number of non-MetS cases of IGT reflects the potential for false reassurance regarding the risk of IGT in an adolescent based on the absence of MetS.th percentile among lean adolescents in NHANES [Interestingly, there appeared to be a sizable number of cases of elevated fasting insulin levels (as an estimate of insulin resistance) in this sample that were not identified by ATP-III MetS or our MetS Z-score. Among adolescents with IGT there was a higher prevalence of elevated fasting insulin—present in 52% of adolescents with IGT—than of ATP-III MetS (24%) or any of the individual components of MetS . There is a clear difficulty in using measures of fasting insulin as an estimate of insulin resistance in settings of glucose excursions, since the mere elevation of BG implies a limitation in secretion of adequate amounts of insulin to normalize BG. Additionally, while fasting insulin correlates highly with other surrogate markers of insulin resistance , it clearly lacks the precision of more robust measures of insulin resistance, such as an insulin clamp ,48. Thusn NHANES  and usedn NHANES -34) may This study had multiple limitations, including the cross-sectional design of NHANES, which limits any conclusions regarding causality. In addition, while NHANES often represents a powerful study, there was only a small subset of adolescents who underwent the OGTT. Finally, we lacked important additional information such as antibodies associated with T1DM as well as genetic information on participants.In conclusion, we found that the presence of MetS and elevated fasting insulin in adolescents had a poor correlation with IGT, an important precursor T2DM as well as a potential finding during the short pre-symptomatic phase of T1DM. This lack of overlap between MetS and IGT may indicate that assessment of MetS is not likely to be a good indicator of which adolescents to screen using an OGTT. These data further underscore the need for further research to assess for other potential contributors to IGT, including T1DM, MODY and polymorphisms associated with poorer beta-cell function. Practitioners should keep in mind other potential causes of IGT, even when evaluating obese adolescents with IGT.ATP-III: Adult treatment panel III; BG: Blood glucose; HDL: High density lipoprotein; IGT: Impaired glucose tolerance; MetS: Metabolic syndrome; MODY: Maturity onset diabetes of the young; NHANES: National health and nutrition examination survey; OGTT: Oral glucose tolerance test; T1DM: Type 1 diabetes mellitus; T2DM: Type 2 diabetes mellitus.The authors have no conflicts of interest to declare.MDD was involved in the design, interpretation and write-up of the study and approved of the final manuscript as submitted. MJG was involved in the design, analysis, interpretation and write-up of the study and approved of the final manuscript as submitted.


Salmonella enterica isolates from human outbreaks or from poultry origin were investigated for their ability to develop direct-tolerance or cross-tolerance to sodium chloride, potassium chloride, lactic acid, acetic acid, and ciprofloxacin after habituation in subinhibitory amounts ( of the minimum inhibitory concentration – (MIC) and of the minimum inhibitory concentration – MIC) of Origanum vulgare L. essential oil (OVEO) at different time intervals. The habituation of S. enterica to OVEO did not induce direct-tolerance or cross-tolerance in the tested strains, as assessed by the modulation of MIC values. However, cells habituated to OVEO maintained or increased susceptibility to the tested antimicrobials agents, with up to fourfold double dilution decrease from previously determined MIC values. This study reports for the first time the non-inductive effect of OVEO on the acquisition of direct-tolerance or cross-tolerance in multidrug-resistant S. enterica strains to antimicrobial agents that are largely used in food preservation, as well as to CIP, the therapeutic drug of salmonellosis.Multidrug-resistant  Salmonella enterica is recognized as the most frequent cause of foodborne disease in the world  is a 2nd generation quinolone, is one of the first-choice agents used to treat salmonellosis. However, a significant rise in the number of Salmonella strains with reduced susceptibility to this antibiotic has been observed in humans and food involved outbreaks  has been cited as a potential compound to control the growth and survival of S. enterica in food  and γ-terpinene (8.3 g/100 g). OVEO solutions (40–0.3 μL mL-1) were prepared in sterile brain heart infusion (BHI) broth  and Tween 80  was added as an emulsifier. Preliminary test were conducted to ensure that the antibacterial activity was because of the OVEO and not Tween 80. The results demonstrated that Tween 80 at the given concentration did not inhibit the growth of the assayed bacterial strains cultivated in BHI broth. Solutions of NaCl (600–50 mg mL-1), KCl (600–50 mg mL-1), AA (160–1.25 μL mL-1), and LA (160–1.25 μL mL-1) were prepared in sterile BHI broth.The antimicrobial agents used in this study were OVEO , CIP , sodium chloride (NaCl P.A.), potassium chloride (KCl), glacial acetic acid (AA), and lactic acid . According to the technical report presented by the supplier, carvacrol is the most prevalent compound in the OVEO assayed in this study (66.1 g/100 mL), followed for S. Enteritidis 209, isolated from feces of outbreak patient, S. Typhimurium 149, isolated from food involved in outbreak and S. Corvallis 297 isolated from poultry (chicken hens). The strains were resistant to aminoglycosides (gentamicin and streptomycin); β-lactams (ampicillin and cefotaxime); quinolones , and sulphametoxazole-trimethoprim. The strains were characterized according to their quinolone resistance mechanism(s) as described in Table 1 , washed twice in sterile saline solution , and resuspended in sterile saline solution to obtain standard cell suspensions at which the OD reading at 660 nm (OD660) was 0.1 , salts (600–50 mg mL-1), acids (16–0.125 μL mL-1), or CIP solutions (0.005 μL mL-1 to 1 μL mL-1) into 90 μL of double-concentration BHI broth in each well. Finally, 10 μL of bacterial suspension (107 CFU mL-1) were added to each well. The microplate was wrapped loosely with cling wrap to prevent bacterial dehydration and ensure that OVEO would not volatilize. Each plate included controls without the antimicrobial test agents. The plates were prepared in triplicate, and they were statically incubated at 37°C for 24 h in a microplate incubator/reader . Following incubation, MIC values were confirmed as the lowest concentrations of OVEO, NaCl, KCl, AA, LA, or CIP at which the OD660 was less than 0.01 . Appropriate amounts of OVEO was added to obtain the desired final concentration ( MIC or MIC), followed by static incubation at 37°C. After 24, 48, and 72 h of incubation, an aliquot of each system was taken  as inoculum (10 μL) to determine the MIC of OVEO, or MIC of NaCl, KCl, AA, and LA using the same microtiter plate assay as described before (P < 0.05) for induction of tolerance were considered when changes in MIC values were equal to or higher than a twofold increase  or heterologous stressing agents . MIC of CIP ranged from 0.12 μL mL-1 to 0.5 μL mL-1, which is considered to be a phenotype of reduced susceptibility to CIP according to the breakpoints designed by S. Corvallis 297. NaCl, KCl, AA, and LA yielded MIC values of 150 mg mL-1, 200 mg mL-1, 2.5 μL mL-1, and 10 μL mL-1, respectively, against all the assayed strains.The MIC values of OVEO against the tested strains ranged from 2.5 μL mLSalmonella strain and the OVEO concentration used for habituation , there was no induction of direct-tolerance in the bacterial cells for 72 h. The maximum decrease in MIC value of OVEO (fourfold double dilution) was observed in S. Corvallis . The decreased MIC values of OVEO against habituated S. enterica was related to the time of exposure to subinhibitory concentrations of OVEO because, with exception of S. Typhimurium 149 habituated to MIC of OVEO, the smallest MIC values were generally found against cells that were pre-exposed to OVEO for 72 h when compared with non-habituated. During all the assessed time intervals, the MIC values of OVEO against non-habituated cells ranged from 5 μL mL-1 to 2.5 μL mL-1.The habituation of the strains to OVEO during the assessed time-intervals  caused a decrease in MIC values of all strains studied. Regardless of the tested Tables 3). There was no effect of time-of-habituation to OVEO on the sensitivity of the habituated cells to NaCl, KCl, and LA. However, the decrease in MIC values of AA against habituated-cells always occurred after 72 h of exposure to subinhibitory concentration of OVEO. MIC values of CIP against the habituated cells were maintained when compared with the previously determined MIC values, indicating that habituation to OVEO did not alter the sensitivity of the strains to CIP. A transient decrease (two-fold double dilution) of MIC values of CIP against S. Enteritidis 209 was observed after 48 h of habituation to MIC and MIC of OVEO.Similar to the direct-tolerance results, MIC values for NaCl, KCl, AA, and LA against the OVEO-habituated cells were the same or decreased (one to threefold double dilution), when compared with MIC values against non-habituated cells  in MIC values of OVEO after habituation for 72 h and was the only strain that exhibited a significant decrease [more than onefold double dilution; Tables 2). The behavior of S. Corvallis could be associated with the low expression of AcrAB-TolC eﬄux pump related genes that has been previously described in this strain  but the sensitivity to NaCl remained unaltered after habituation  in MIC values of gentamicin, erythromycin, chloramphenicol, tetracycline, streptomycin, and trimethoprim .To our knowledge, this is the first study that investigated the tolerance development by multidrug-resistant The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.